Fiber sampler for recovery of bioaerosols and particles

ABSTRACT

A bioparticle collection device and an aerosol collection system. The bioparticle collection device includes a collection medium including a plurality of fibers formed into a fiber mat and configured to collect bioparticles thereon, and includes a viability enhancing material provider disposed in a vicinity of the plurality of fibers and configured to provide a viability enhancing material to the collected bioparticles to maintain viability of the bioparticles collected by the fiber mat. The aerosol collection system includes an aerosol pumping device configured to entrain particles in an gas stream, an aerosol saturation device configured to saturate the particles in the gas stream with a biocompatible liquid, and an aerosol collection medium downstream from the aerosol saturation device and including a plurality of fibers formed into a fiber mat for collection of the saturated aerosol particles.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is related to and claims priority under 35 U.S.C.119(e) to U.S. Application Ser. No. 61/374,466, filed Aug. 17, 2010,entitled “Fiber Sampler for Recovery of Bioaerosols and Particles,” theentire contents of which are incorporated herein by reference.

This application is related to U.S. application Ser. No. 11/559,282,filed on Nov. 13, 2006, entitled “Particle Filter System IncorporatingNanofibers,” the entire contents of which are incorporated herein byreference. This application is related to U.S. application Ser. No.10/819,916, filed on Apr. 8, 2004, entitled “Electrospinning of PolymerNanofibers Using a Rotating Spray Head,” the entire contents of whichare incorporated herein by reference. This application is also relatedto U.S. application Ser. No. 10/819,942, filed on Apr. 8, 2004, entitled“Electrospray/electrospinning Apparatus and Method,” the entire contentsof which are incorporated herein by reference. This application isrelated to U.S. application Ser. No. 10/819,945, filed Apr. 8, 2004,entitled “Electrospinning in a Controlled Gaseous Environment,” theentire contents of which are incorporated herein by reference. Thisapplication is related to U.S. Ser. No. 11/130,269, filed May 17, 2005entitled “Nanofiber Mats and Production Methods Thereof,” the entirecontents of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under HSHQDC-09-C-00154awarded by DHS. The government has certain rights in the invention

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is related to fibers, methods, and devices for collectionof bioaerosols and particles on fiber structures. The invention is alsorelated to electrospun materials for filtration and air sampling, inparticular the collection of bioaerosols.

2. Description of the Related Art

Collection of both indoor and outdoor air samples is important formonitoring air quality. A wide range of microorganisms are of interestincluding bacteria, fungi and viruses. From a health standpoint, toxinsand allergens may be of interest as well. For example see, J. M. Macher(1999) Bioaerosols, Assessment and Control, American conference ofGovernmental Industrial Hygienists, Cincinnati, Ohio.

More recently, concerns about airborne pathogens being present due tonatural processes, accidents, or terrorist attacks has led to the needfor improved sampling systems. In addition to the problem of collectingthe aerosol (particles) is the problem of recovering the particles foranalysis. In the case of biological particles, a common problem is thatthe organisms die during collection or after collection while awaitinglaboratory analysis. Current sampling methods onto microbiological mediado not permit extended sampling times beyond 30-45 minutes in the casewhere preservation of viable organisms is of interest.

In general, a concentrated, viable collect of submicrometer biologicalparticles has been recognized in the art as a challenge. Each bioaerosolsampling method has limitations with respect to sampling time,desiccation, shelf life of sample, complexity, compatibility withanalysis via PCR and live recovery. Some evaluations are given byGriffiths and Decosemo (1994); Henningson and Ahlberg (1994); Wang,Reponen et al. (2001); Tseng and Li (2005); Verreault, Moineau et al.(2008); Mainelis and Tabayoyong (2010) listed below:

-   Griffiths, W. D. and G. A. L. Decosemo 1994. The Assessment of    Bioaerosols—a Critical-Review. Journal of Aerosol Science 25(8):    1425-1458.-   Henningson, E. W. and M. S. Ahlberg 1994. Evaluation of    Microbiological Aerosol Samplers—a Review. Journal of Aerosol    Science 25(8): 1459-1492.-   Mainelis, G. and M. Tabayoyong 2010. The Effect of Sampling Time on    the Overall Performance of Portable Microbial Impactors. Aerosol    Science and Technology 44(1): 75-82.-   Tseng, C. C. and C. S. Li 2005. Collection efficiencies of aerosol    samplers for virus-containing aerosols. Journal of Aerosol Science    36(5-6): 593-607.-   Verreault, D., S. Moineau and C. Duchaine 2008. Methods for sampling    of airborne viruses. Microbiology and Molecular Biology Reviews    72(3): 413-444.-   Wang, Z., T. Reponen, S. A. Grinshpun, R. L. Gorny and K.    Willeke 2001. Effect of sampling time and air humidity on the    bioefficiency of filter samplers for bioaerosol collection. Journal    of Aerosol Science 32(5): 661-674.

The collection of bioaerosols is currently performed by a number ofdevices that have been available for quite some time. Common bioaerosolsampling devices include:

-   -   Impactors where a jet of air deposits the bioaerosol particle on        a media surface.    -   Impingers where the jet of air impinges on a surface within a        liquid filled container.    -   Filters where the particles are collected on the surface of the        filter.

Impactors are limited with respect to sampling time because thecollection media used to enumerate the number of colonies of organismsfor viability after collection is subject to desiccation, thus limitingthe sampling time. Also typical impactors designed for microorganismshave a lower particle size collection limit of about 0.5 micrometers.(Anderson, A. A. (1958) New sampler for collection, sizing, andenumeration of viable airborne particles, J. Bacteriol. 76, 471-484)

Impingers are limited in their sampling time from the evaporation of thecollecting fluid. The collection efficiency is dependent on the volumeof fluid in the impinger. Also the microorganisms may be lost byreaerosolization from the fluid during sampling (Grinshpun, S. A., K.Willeke, V. Ulevicius, A. Juozaitis, S. Terzieva, J. Gonnelly, G. N.Stelma and K. P. Brenner (1997) Effect of impaction, bounce andreaerosolization on the collection efficiency of impingers. Aerosol Sci.Technol. 26, 326-342.).

Filters and other collection media such as membranes have long been usedto trap aerosol and bioaerosols for subsequent analysis thereof. Filterswith a poor figure of merit or quality at least require higher pressuresto force air flow through. An example consequence is that in portablesamplers operation is severely limited due to battery life in thesamplers with filters with high pressure drop. Filter figure of merit orquality is defined as FoM=−log (Pt)/ΔP, where Pt is the penetration ofparticle at a specific size through the filter and ΔP is the pressuredrop at a specific gas flow rate. The larger the FoM, the better will bethe performance of the filter. See Hinds, W. C. (1982) AerosolTechnology, Wiley, New York, N.Y.). Further, the flow of air through thefilters or membranes after a biological aerosol has been trapped canlead to the desiccation of the medium about the bioaerosol and death ofthe bioaerosol.

Thus, in general, a list of existing air sampling technologies forbioparticles and their drawbacks are provided below.

Typical longest Sampler d₅₀ sampling time Notes Impinger ~0.3 μm 30 minGood for short e.g. AGI-30 term sampling Impactor ~0.7 μm 20 minCollection on e.g. Anderson agar reduces desiccation SKC ~0.3 μm  8 hrsFluid for long BioSampler term sampling interferes with PCR Filtration *60 min^(†) Desiccation is a e.g. 37-mm significant cassette with problemwith Nucleopore filtration * Filtration has a most penetrating sizeabout 0.1 to 0.3 μm with efficiency of collection typically high (>80%)across size range. ^(†)Longer term sampling is possible but organisms donot survive.

SUMMARY OF THE INVENTION

In one embodiment of the invention, there is provided a bioparticlecollection device including a collection medium including a plurality offibers formed into a fiber mat and configured to collect bioparticlesthereon, and includes a viability enhancing material provider disposedin a vicinity of the plurality of the fibers and configured to provide aviability enhancing material to the collected bioparticles to maintainviability of the bioparticles collected by the fiber mat.

In one embodiment of the invention, there is provided an aerosolcollection system including an aerosol pumping device configured toentrain particles in a gas stream, an aerosol saturation deviceconfigured to saturate the particles in the gas stream with abiocompatible liquid or vapor and an aerosol collection mediumdownstream from the aerosol saturation device. The aerosol collectionmedium includes a plurality of fibers formed into a fiber mat forcollection of the saturated aerosol particles and a viability enhancingmaterial provider disposed in a vicinity of the plurality of fibers andconfigured to provide a viability enhancing material to the collectedbioparticles to maintain viability of the bioparticles collected by thefiber mat.

In one embodiment of the invention, there is provided a method forcollecting aerosols. The method entrains particles in an gas stream,saturates the particles in the gas stream with a biocompatible liquid,and collects the saturated aerosol particles in a filtration mediumincluding a plurality of fibers formed into a fiber mat and a viabilityenhancing material provider disposed in a vicinity of the plurality offibers and configured to provide a viability enhancing material to thecollected bioparticles to maintain viability of the bioparticlescollected by the fiber mat.

In one embodiment of the invention, there is provided a bioparticlecollection device including a collection medium including a plurality offibers formed into a fiber mat and an osmotic material disposed incontact with the plurality of fibers.

In one embodiment of the invention, there is provided an aerosolcollection system including an aerosol pumping device configured toentrain particles in a gas stream, an aerosol saturation deviceconfigured to saturate the particles in the gas stream with abiocompatible liquid, and an aerosol collection medium downstream fromthe aerosol saturation device. The aerosol collection medium includes aplurality of fibers formed into a fiber mat for collection of thesaturated aerosol particles and an osmotic material disposed in contactwith the plurality of fibers.

In one embodiment of the invention, there is provided a method forcollecting aerosols. The method entrains particles in a gas stream,saturates the particles in the gas stream with a biocompatible liquid,and collects the saturated aerosol particles in a filtration mediumincluding a plurality of fibers formed into a fiber mat, and an osmoticmaterial disposed in contact with the plurality of fibers.

It is to be understood that both the foregoing general description ofthe invention and the following detailed description are exemplary, butare not restrictive of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 is a table showing sampling challenges for the sampling andpreservation of bioaerosols;

FIGS. 2A and 2B are schematics of fiber structures of the invention;

FIG. 3 is a schematic of a condensation growth tube with impaction ofdroplet encapsulated particles on a fiber substrate of the invention;

FIG. 4 is a schematic of a combination of the condensation growth tubeand a fiber filter where the particles are removed by filtration;

FIG. 5 is a schematic showing the collection of bioaerosol particles ina nanofiber filter;

FIG. 6 is a schematic showing a combination of a humidifying sectionfollowed by a fiber filter of the invention;

FIG. 7 is a schematic showing the fiber filter with injection of waterinto the fiber filter to maintain an environment on the filter of 70% RHin this one illustrative example;

FIG. 8 is a schematic of applying fiber collection surfaces tobioaerosol collection with an impactor;

FIG. 9 is a schematic of the combination of humidification of thebioaerosol with an impactor containing fibers on the collection surface;

FIG. 10 is a schematic showing a cascade impactor with fiber collectionsurfaces and water introduction to maintain a controlled relativehumidity;

FIG. 11A is Table 1 depicting an example of polymer and surfacechemistries studied in this invention;

FIG. 11B is a SEM micrograph of a perspective view of a nanofiberstructure formed by deposition of PU on a part of a web material;

FIG. 11C is another SEM micrograph of a perspective view of a nanofiberstructure formed by deposition of PU on web material showing nanofibercoverage and orientation over an opening in the underlying web material;

FIG. 11D is another SEM micrograph showing a cross section of ananofiber structure formed by deposition of PU on web material;

FIG. 11E is another SEM micrograph of a nanofiber structure formed bydeposition of PU on web material;

FIG. 11F is a composite view showing differently scaled depictions of ananofiber sampling filter in a 37 mm cassette format;

FIG. 12A is a composite of two scanning electron micrograph SEM imagesshowing a collection including a Bacillus globigii (Bg) spore and whatare likely MS2 virus particles;

FIG. 12B is a graph of pressure drop curves (pressure drop versus facevelocity) for two common commercial air sampling filter materials ascompared with nanofiber filter media composed of PSU or PU depositedthereon;

FIG. 13 is a bar graph depicting colony forming units per liter of airsampled for a challenge of Bacillus for different sampler configurationsand substrates;

FIG. 14 is a bar graph depicting colony forming units per liter of airsamples for Pseudomonas for different sampler configurations andsubstrates;

FIG. 15 is a bar graph depicting plaque forming units per liter of airsamples for bacteriophage MS2 for different sampler configurations andsubstrates;

FIG. 16 is Table 2 depicting the results of the collection of Bg CFUsand MS2 PFUs by condensation growth tube (CGT) with impaction onto PUnanofibers, the All-Glass Impinger (AGI), and an aerodynamic particlesizer (APS);

FIG. 17 is Table 3 depicting a comparison of the fiber filter mats ofthe invention to a standard Teflon filter using the virus MS2;

FIG. 18 is a comparison of viabilities between fiber filter mats of theinvention and gelatin and Teflon filters where a bioaerosol of Serratiawas sampled for 3 hours;

FIG. 19 is a comparison of viabilities between fiber filter mats of theinvention and gelatin where a bioaerosol of Serratia was sampled, andshows the impact of sampling face velocity on the viability of Serratia;

FIG. 20 is a depiction showing of the viabilities obtained whencollecting fragile Yersinia rohdei using RH controlled filtration withthe fiber filter mats of the invention;

FIGS. 21A and 21B are Tables 4 and 5 depicting organism survivability ondifferent surfaces and different environmental conditions;

FIG. 22 is a depiction showing the storage of the slightly fragileStaphylococcus and very fragile organism Yersinia at different storageconditions;

FIG. 23 is a schematic depiction of a sample storage deviceincorporating a moisture providing material; and

FIG. 24 is a schematic depiction of another sample storage deviceincorporating a moisture providing mechanism.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “bioparticles” means microbes and other biologicalparticles such as for example bacteria, viruses, and biologicallyderived particles such as proteins, cell fragments, etc.

As used herein, “viable” or “viability” is defined as the capability ofhaving a collected organism becoming active again after being placedinto a favorable environment. For example, a collected bacteria spore orvegetative bacterium being placed into a growth media and incubatedunder appropriate conditions for growth resulting in growth andreproduction of the organism. For example, a collected virus beingexposed to its desired host and incubated under appropriate conditionsresulting in the virus infecting the host.

As used herein, “collection viability” means the capability to keep apercentage of bioparticles in a collection medium of this inventionalive during the collection event.

As used herein, “storage viability” means the capability to keep apercentage of bioparticles in a collection medium of this inventionalive from the time of collection until the bioparticles are analyzed orcounted.

As used herein, “viability enhancement” or “enhanced viability”encompasses both collection viability and storage viability and meansthe capability to collect a percentage of bioparticles from a mediumwithout death and keep the collected bioparticles alive until thebioparticles are analyzed or counted.

As used herein, an “osmotic material” is as any material that has thecapacity to provide transport of liquids (such as for example water ornutrients) to or from the collected bioparticles. For example, a fibercomposed of a hydrophilic polymer would represent on one kind of osmoticmaterial.

As used herein, “design limiting” organisms are organisms which areextremely fragile and extremely difficult to keep alive.

In order to determine if an organism is infectious for the purpose ofmaking health related decisions, the viability must be assessed byculture methods where the presence of live organisms at the start of theculture is needed.

Maintaining viability during and after collection is well known to be achallenge. Some organisms are very hardy, such as bacterial spores.These organisms can be very difficult to kill. The same traits that makethem difficult to kill make them more readily kept alive or viableduring collection and storage. Other organisms are extremely fragile andextremely difficult to keep alive. Maintaining viability of these designlimiting microorganisms during collection and during storage is achallenge. Organisms lose viability during collection due to desiccationby either the air moving past these organisms during the collectionprocess or from a process such as evaporation. Also, any condition thatleads to an increase in hydroxyl radicals will decrease viability.

Thus, collection of bacteria and virus (microbes) while keeping thesebioparticles viable in the case of long term sampling is problematic.

Referring now to the drawings, wherein like reference numerals designateidentical or corresponding parts throughout the several views, FIG. 1details the challenges overcome by the invention for representativeorganisms important from a health standpoint. More specific, challengesdetermined by the inventors for conducting long term air sampling, butare not restricted to long term air sampling, in the collection ofviable bioparticles include:

-   -   Viability during sampling    -   Sampling duration time    -   Viability of collected sample during storage    -   Compatibility of collected sample on the collection medium with        analysis techniques.

Bioparticle Sample Collection Devices

Electrospun micro and nanofibers from polymer solutions provide a highsurface area environment with tunable surface chemistries which can beconducive to the collection and retention of biological particles.Indeed, the invention in one embodiment provides a sampling device forthe collection and recovery of particles, including biological particlessuch as bacteria, viruses, and yeasts. The sampling device provides forenhanced viability of biological particles and provides for quantitativerecovery of samples for laboratory analysis, as detailed below.

The invention provides for a device, based on a fiber mat or a nanofibermat that provides for collection of bioparticles including bacteria,fungi, viruses, and other biological particles (e.g., bioaerosols). Thecollection is achieved in one aspect of the invention either through theuse of the fiber mat as a filter, for example a high efficiency lowpressure drop nanofiber flow-through filter, through the use of thefiber mat as a substrate for impaction of particles, or for the use of afiber mat as a wipe.

In one embodiment of the invention, the bioparticles are kept viable forextended periods of time (e.g., 1 day to >7 days) without extraordinaryefforts because the biological particles are collected in amoisture-rich (or nutrient-rich) fiber mat or nanofiber filter mat.Furthermore, samples can be recovered from the mats for analysis byextraction in buffer or other suitable liquid. Alternately, the fiberscan be configured to be dissolved using, for example a low acid orenzymatic solution. Indeed, in one embodiment of the invention, the nanoor microfiber material can be constructed from polymers that provide fordissolution in water or an appropriate buffer. Such capability canimprove recovery of collected bioparticles for culture and non-cultureanalysis method such as PCR (polymerase chain reaction), ELISA(Enzyme-Linked Immunosorbent Assay), and a variety of other molecularand biochemical techniques.

In one embodiment of the invention, the fibers are deposited on avariety of backing materials which could include moisture absorbingproperties or ability to provide moisture to the fiber mat; for example,super-soaker polymers, hydrophilic polyurethane foam, blotter paper,polymer nonwoven mats containing hydroscopic salts such as lithiumchloride, and related methods. Accordingly, the fibers of the inventioncan form in one embodiment a bioparticle collection device including acollection medium including a plurality of fibers formed into a fibermat and an osmotic material disposed in contact with the plurality offibers.

In one embodiment of the invention, the structure and surface chemistryof the fibers, incorporation of additives, or mixed fiber materialsincorporating osmotic materials can contribute to the collection andpreservation of the bioparticles. Furthermore the container or packagingof the fiber material can aid in preservation. For example, a sealedcontainer containing a hydrogel or other material can be used tomaintain RH to aid in viability preservation.

U.S. Pat. No. 4,805,343 (the entire contents of which are incorporatedherein by reference) describes for example cellulose acetate hollowfibers that have osmotic properties. Such fibers (or other hydrophilicfibers) could be used in the present invention to provide an externalsupply of water or nutrients transported to the fiber mats collectingthe bioaerosols. Alternatively, cellulose acetate fibers could beintermixed into the fiber mats collecting the bio-aerosols.

The use of a fibrous matrix to collect and preserve the bioparticlesalso provides advantages from the equipment design and operation pointof view. A long term (>8 hrs) liquid-based sampler typically wouldrequire a fluidics system to remove sample and replenish buffers. AnRH-controlled fibrous material format would not require as an extensivefluidics system. Furthermore, if a large amount of dust, pollen, andother small particles are present in the fluidics system, then theinstrument could become clogged.

A fibrous matrix approach that is free of a fluidics system couldtolerate samples laden with dust, but these particles would not shut thesystem down. Additionally, the mass of liquid needed to operate a systemlong term could be significantly less. The weight and complexity of alow liquid use or nearly liquid-free sample collection/preservationsystem could also be much less compared to a liquid collection system.

In one embodiment of the invention, the fibers are deposited on variousbacking materials, and the combination of the fibers and the backingmaterials is used as an impaction substrate for collection of theaerosol. For example, the fibers can be electrospun onto a foil andplaced as a part of the impaction plate in a standard impactor for airsampling.

The fiber matrix (and especially a nanofiber matrix) provides a highsurface area environment for collecting organisms. At the micro-scale ofbacteria and viruses, surface chemistry can be important. Using polymersprovides for adjustable surface chemistries from hydrophilic tohydrophobic. Furthermore, hydrogels including polymer networks thatreadily hold water can be used to regulate the moisture content of thenanofiber matrix. Examples of such systems include polymers of acrylicacid combined with sodium hydroxide and co-polymers of poly(2-hydroxyethyl methacrylate) (polyHEMA). Complex multi-fiber and layeredstructures can easily be fabricated to provide a mixed environment thatcannot be obtained with a liquid system. This mixed environment canpotentially provide a way for a variety of organisms that preferdifferent environmental conditions to exist in the same sample. Anexample of a mixed environment is simultaneously electrospinning twodifferent polymers onto a common collection substrate thus creating afibrous mat with two different polymers which would have two differentsurface chemistries and/or fiber diameters.

In one embodiment of the invention, the fibrous matrix sample collectiondevice includes mechanisms such as those described above or othermechanisms to provide moisture or to maintain the RH in a desired range,for example from 65% to 85% or more precisely 70% to 85% or moreprecisely 75% to 81%.

In one embodiment of the invention, a polyurethane PU fiber, thestructure of the PU nanofibers, the corresponding nonwoven, and the RHall contribute to viability maintenance. In this embodiment, theviability enhancing aspect appears to be only the PU nanofiber mat andsurface humidity of the nanofibers, and there is no need for anadditional osmotic material, although such an addition could be used.

In one embodiment of the invention, the sample collection deviceprovides viable storage at ambient temperature and RH. In anotherembodiment of the invention, viability maintenance is enhanced,especially for particularly fragile organisms, via storage at cooledconditions. Storage of fragile organisms such as Yersinia has beendemonstrated for more than 9 days when stored on a polyurethane (PU)nanofiber media in a laboratory refrigerator.

On one hand, while keeping collected organisms wet may result ingermination or growth, and the collection conditions might be good forone class or organisms, the collection conditions might be bad foranother class of organisms. On the other hand, an overly dry environmentcan also kill organisms. A fibrous matrix (optionally combined withother humidity control devices) can provide a relative humidity(RH)-controlled environment to improve preservation of viability ofbioaerosols while potentially simplifying sample handling and storage.

Filter Collection Systems

Nanofibers can be used in one embodiment of the invention as a lowpressure drop, high efficiency collection filter in any standardsampling form such as the commercial ‘37 mm air monitor cassette’ orother sampling cassette device. (The nanofibers can also be used in animpaction device for example in an eight-stage impactor.)

FIGS. 2A and 2B show schematics of various fiber structures. Thedifferent fibers designations indicate different function inmicroorganism preservation. In one embodiment, fibers are present thatcontain or regulate water moisture. For example, the fibers such ashydrogel polymers or crosslinked polyHEMA or gelatin or similar suchmaterial can be used as for hydration. Alternate versions includehydrophilic polymers, like cellulose and its derivatives (e.g. celluloseacetate) and may include incorporation of hydroscopic salts such aslithium chloride; or hydrogel particles, such as those formed fromacrylic acid combined with sodium hydroxide, with these particlesentrapped in the fiber matrix. In FIGS. 2A and 2B, the white spacebetween the two designated types of fibers represents air space or otherfibers in the collection mat (for example having a density and size topromote collection viability and storage viability of a bioaerosol).

In one embodiment, the white space between the two designated types offibers may be filled with particles which themselves contribute to theviability of the collected bioaerosols. These particles can beintroduced during the electrospinning process in a manner as describedin U.S. Pat. No. 7,297,305 (the entire contents of which areincorporated herein by reference). For example, particles (e.g.,antioxidant particles or nutrient particles) which can contribute to theviability of the collected bioaerosols can be introduced into the fluidssuitable for electrospraying and/or electrospinning. Alternatively,these particles can be introduced in a manner as described in U.S. Pat.Appl. No. 2006/0264140 (the entire contents of which are incorporatedherein by reference).

In this process, particles which can contribute to the viability of thecollected bioaerosols are delivered into a fiber-extraction region of anelectrospinning apparatus. The introduced particles collide and combinewith the electrospun fiber material during formation of the fibers andthe fiber mat. Alternatively, these particles can be introduced afterthe electrospinning process by flowing a solution (non-reactive with thefibers in the fiber mat and containing the particles of interest)through the fiber mat. The solution can be thereafter evaporated orretained if the solution itself is a substance which can contribute tothe viability of the collected bioaerosols.

The fibers in FIGS. 2A and 2B may be aligned or may have randomorientations. The fibers in FIGS. 2A and 2B would in one embodiment bein contact with one another in the fiber mat.

In one embodiment of the invention, the hydration fibers are not berequired. In one embodiment of the invention, the preservation fibersare not required. When used, the preservation fibers, due to theirsurface chemistry and structure, promote preservation of thebioparticles. A more detailed description of preserving fibers isprovided below.

Accordingly, FIG. 2A illustrates intermixed fiber material made bysimultaneous electrospinning onto a common collection plane, and FIG. 2Billustrates the concept of a layered structure that can be formed eitherby sequential electrospinning to make a layered structure or by spinningfrom opposing directions to a common plane to simultaneously build totwo sides of the composite, layered structure.

As noted above, the fiber mat of the invention can be configured as animpaction substrate or as a flow though filter, and can be used in avariety of air sampling systems and configurations.

Methods of Conditioning Bioparticle prior to Collection

In one aspect of the invention, the conditioning of inlet air containingbioparticles facilitates the collection of viable bioparticles. In oneaspect of the invention, the collection of the bioparticles occurs ontoan appropriate substrate (media) that aids in collection of viablebioparticles, aids in storage of the viable bioparticles, and permitsanalysis via a variety of techniques (e.g. live culture, PCR-basedanalysis methods, immuno-based assays, etc.).

FIG. 3 is a schematic of a condensation growth tube with impaction ofdroplet encapsulated particles on a fiber substrate of the invention.The fiber substrate provides an environment for preserving microorganismviability during sampling and storage. U.S. Pat. No. 6,712,881 (theentire contents of which are incorporated herein by reference) depicts awater condensation growth tube (CGT) technology where particles areenlarged by water condensation. The air containing bioparticles andother particles flow through the condenser having a second temperaturegreater than the flow temperature and a vapor pressure of condensingvapor at the walls of the condenser near saturation. This technology iscapable of condensationally enlarging particles as small as viruses(0.01 μm-0.02 μm) into 2-μm diameter water droplets while maintaining alaminar flow. After the droplets have been formed, the encapsulatedparticles can be focused and collected inertially at low velocities,reducing the potential damage to the microorganism and minimizing theenergy and noise associated with the pump.

In one embodiment of the invention, exit jets from a CGT device canimpact bioaerosol or other aerosol particles onto a fiber substrate ofthe invention. In one embodiment of the invention, in the CGTconfiguration, small particles (e.g., sub-micron sized particles) areenlarged through condensational growth while still airborne, essentiallyencapsulating each particle within a micrometer-size (or larger)moisture droplet that is readily collected at low velocity onto thefibrous or nanofibrous mat.

FIG. 4 is a schematic of a combination of the condensation growth tubeand a fiber filter where the particles are removed by filtration. Inthis arrangement, the condensation growth tube is coupled with a fiberfilter. The particles are removed by the filtration mechanisms ofimpaction, interception, and diffusion. In one embodiment of theinvention, the exit gas from the CGT flows through a fiber filter. Inone embodiment of the invention, in the CGT, small particles (e.g.,sub-micron sized particles) are enlarged through condensational growthwhile still airborne, essentially encapsulating each particle within amicrometer-size (or larger) moisture droplet that is readily collected.

Accordingly, in this technique of the invention, a bioparticle isexposed to the vapor or a working fluid (for example biocompatiblefluids such as water) in a saturation chamber. Subsequently, vaporcondensation onto bioparticles is induced by either adiabatic expansionor cooling in the condensing chamber, or by mixing with a coolerairflow.

With the CGT, sub-micron particles (including bioparticles) can be grownusing a supersaturated vapor to a size where collection on the fibermats or nanofiber mats of the invention is enhanced in terms of particleentrapment and bio-particle survivability. Accordingly, in oneembodiment of the invention, the formed particle-water-condensatebioparticles are collected on the collection medium of the invention.

FIG. 5 is a schematic showing the collection of bioaerosol particles ina fiber filter. The collection in the fiber filter occurs byinterception, impaction, and diffusion. In one embodiment, a nanofiberfilter has low pressure drop and high efficiency and creates anenvironment for preservation of microorganisms.

FIG. 6 shows a fiber filter following a humidifying section whichcontrols the humidity at the fiber filter at a target value or range,for example 50 to 85% RH. The humidification chamber (in one embodiment)is disposed at the site of the mixing of humidified air with thebioaerosol sample. The humidification chamber (in another embodiment) isa chamber where water is introduced into the air by wetted porous wallsto maintain e.g., a relative humidity of 70 to 80% at the filter.

FIG. 7 shows the arrangement of introducing water into the fiber filterto maintain an environment e.g. a relative humidity of 70 to 80% whichpreserves microorganisms during sampling. The humidity wouldalternatively be maintained during storage.

FIG. 8 shows combination of the fiber with a cascade impactor. Thefibers, including nanofibers, prevent rebound of the particles and canprovide an environment to preserve the microorganisms. The impactorshave small holes forming jets of air directing particles at thecollection stage at a high velocity (usually less than 0.3 Mach). Theinertia of the particles causes the particles to impact on the fibers.

FIG. 9 shows an impactor containing fiber collection on collectionstages with a humidification of the bioaerosol. Humidification (in oneembodiment) involves the mixing of the air containing bioaerosol withmoist air or (in another embodiment) evaporation of water within thehumidification chamber from wet porous walls.

FIG. 10 shows the introduction of water into an impactor with fiber onthe collection surface to maintain a controlled humidity e.g., at 70%RH.

Accordingly, in this invention, there are provided a number of ways forconditioning of bioparticles prior to collection which include use of acondensation growth tube, adding water moisture to the sampled airstream, and regulating the relative humidity (RH) of the sampled airstream. Addition of water moisture or regulation of the RH can beachieved via a number of methods including use of a wet walled tube toprovide humidity to the sampled air, atomization of water to providehumidity to the sampled air, mixing a wet or dry air stream with sampledair stream to provide air stream at target RH (wet air could begenerated through bubbling air through water, a wet walled tube,atomization of water, etc.), and other ways to regulate the RH of asampled air stream.

Methods of Making Fiber Substrates for Bioparticle Collection

Electrostatic spinning of polymer solutions to form micro and nanodiameter fibers, better known as electrospinning, is a ready method tomake nonwoven fibrous mats. In one embodiment of this invention,electrospinning is used to make fibrous mats but other methods offabricating mats of micro and nanofibers may also be a route to formfibrous structures described in this invention. U.S. Pat. Nos. 5,494,616and 6,520,425; and Badrossamy M R et al., Nano Letters 2010, 10(6):2257both describe alternative techniques applicable to the invention. Theentire contents of these documents are incorporated herein by reference.

A wide variety of polymers can be electrospun into fibers including bothsynthetic polymers such as polystyrene and natural polymers such ascollagen and gelatin. Polymers offer hydrophobic to hydrophilic surfaceproperties including functionalities similar to sugars or proteins. FIG.11A shows Table 1 is an example of only a limited number of polymer andsurface chemistries that are suitable for this invention.

In terms of the use of electrospun fibers for filter mats suitable forthe invention, U.S. Pat. Appl. Publ. No. (2005/0224999), the entirecontents of which are incorporated herein by reference, describes theuse of an electronegative gas to facilitate the electrospinning processby the introduction, for example, of carbon dioxide (CO₂) around thespinning orifice or emitter. Gases such as CO, CF₄, N₂O, CCl₄, CCl₃F,CCl₂F₂ and other halogenated gases can be introduced into theelectrospinning environment. These electronegative gases stabilize theTaylor cone formed by the polymer jet as it comes off the needle,reduces corona discharge at the needle, and reduces fiber diameter.Furthermore, spinning in a controlled environment ensures lesscontamination of the fibers, improves safety, and adds another dimensionof control parameters that can be used to fine-tune fiber formation.

An electronegative gas can be passed coaxially with the spinning needlealong with use of a controlled gas environment. Typically, a gas shroudis used to provide the coaxial gas flow. A typical shroud can be in theshape of an annulus having an outside radius of about 0.48 cm and aninside radius of about 0.40 cm. Insulating and metallic shroud memberscan be used. A variety of geometries and sizes are possible; such as forexample a circular outside with a hexagonal inside being an additionalgeometry. In the annular geometry, a distance from an exit end of theannulus where gas is emitted to the tip of the electrospinning elementcan range from flush (0 cm) to 8 cm; with a typical distance beingaround 4 to 5 cm, and with the distance being 4.7 cm for the detailedexamples later.

Control of the electrospinning conditions has produced polymernanofibers with an average fiber diameter AFD of 100 nm and less.Nanofibers less than 400 nm have been found to improve the filtrationproperties of the resultant fiber when combined with other elements ofthe invention.

Additives in the polymer solution can make a substantial difference infiber size and quality. Addition of trace amounts of a salt or asurfactant increases the solution conductivity and hence the chargeaccumulation at the tip of the electrospinning element resulting inlarger stretching forces applied to the forming fiber, hence smallerdiameter fibers. The surfactant also reduces the surface tension of thepolymer allowing for even smaller fibers to be spun. Lithium salts, (forexample, lithium chloride and lithium triflate) or surfactants such astetra butyl ammonium chloride (TBAC) are suitable for the invention.Lithium salt concentrations from 0.01 to 3 wt % are suitable for theinvention. Concentrations of TBAC of between 0.06 and 0.4 wt %, wereexemplary, although other concentrations are suitable.

Stainless steel extrusion tips from 0.15 mm to 0.59 mm internaldiameters (ID) are suitable for the invention. Larger and smallerdiameters may also be used. Teflon™ capillary tubes with ID from 0.076mm to 0.31 mm are suitable for the invention. Larger and smallerdiameters may also be used. Both types of orifices can produce smallfibers. For both orifices, low flow rates of the polymer solution (e.g.,0.05 ml/hr) coupled with high voltage drops typically resulted in thesmallest fiber diameters (e.g., AFD less than 100 nm). In both cases,the voltage was set to 22 kV to 30 kV for a 17.8 cm to 25.4 cm gap(i.e., distance between emitter 2 and mesh 7). Of note is that thevoltage per electrospinning-gap is one parameter determining the pullingstrength; this gap also determines a travel time thus partly determiningfiber stretching time.

Besides stainless steel and Teflon™ extrusion tips, in the invention,other materials (provided the materials are non-reactive with thesubstance being electrospun including any solvent used in theelectrospinning process) can be used such as for example polymers,glass, ceramic, or metal extrusion tips.

The relative humidity RH of the electrospinning chamber effects fibermorphology. For example, when using 21 wt % PSu (M_(w)˜35,000 g/mol) inDMAc, a high RH (e.g., >65%) resulted in fibers that have very fewdefects and smooth surfaces but larger diameters. A defect in a fiber isin general seen as a deviation from a smooth round fiber of long length.Defects thus are beads on the fiber, variations in fiber diameter in theaxial direction, etc. A low RH (e.g., <13%,) resulted in smaller fibersbut more defects. Modestly low RH (e.g., 40% to 20%) typically producedsmall fiber size with fewer defects.

A variety of mechanisms are suitable in the invention to control thechamber RH such as placing materials that absorb (e.g. calcium sulfate)or emit water moisture (e.g., hydrogels), operating a small humidifierin the chamber, and adding moisture into the process gas streams priorto introduction to the electrospinning chamber. For example, positiveresults were obtained by bubbling CO₂ through deionized (DI) water andthen introducing the humidified CO₂ gas into the chamber. In oneembodiment of the invention, two gas streams (e.g., one humidified andone dry) are used to obtain a desired RH for the chamber and/or for thegas jacket flowing over the electrospinning orifice.

The fiber diameter obtained in the invention is a function of thepolymer molecular weight, the polymer architecture, the solvent orsolvents, the concentration of polymer in the solvent system, theadditives and their concentration, the applied electrospinningpotential, the gap between the spinning orifice and ground, the size andshape of the spinning orifice, the polymer solution flow rate, the flowrate and composition of the process gas that flows over the needle, theRH of the process gas, and the partial pressure of the solvent(s).

Other embodiments of the invention could use different polymer solventsystems and hence different electrospinning conditions to obtainappropriate nanofibers. Furthermore, the same polymer solvent systemscould be combined with different electrospinning conditions to createimproved fibers or fibers tailored for alternative applications. Forexample, the jacket of CO₂ gas flowing over the needle could containsolvent vapor in order to lower the evaporation rate of the solvent(s)in the polymer jet formed at the needle tip, thus increasing stretchingtime of the polymer fiber. The partial pressure of the solvent can alsobe modified via control of temperature, pressure, and mixture ofsolvents. The solvent concentration as determined by a relativeconcentration in the atmosphere is controlled to between 0 and 100%.

Filter Support Structures

In addition to obtaining nanofibers having few defects and a closedistribution in fiber diameter sizes, the construction of a support andpreparation of the surface of the support affect the resultant fiber matand the resultant filter properties. In one embodiment of the invention,a macroscopic mesh provides adequate support for the nanofibers towithstand the forces exerted on filter mat during filtration andcollection of biological medium. The support mesh contributes minimallyto pressure drop of the resultant filter.

Filters formed with rigid meshes that contained 1.27 cm, 0.635 cm, or0.159 cm (i.e., American Engineering standard sizes: ½″, ¼″ and 1/16″respectively) openings using copper, brass, nickel, stainless steel, andaluminum metal are suitable for the invention. Smaller sizes have alsobeen found acceptable including meshes with openings as small as 0.031cm. Aluminum window screen with openings about 1.2 mm×1.6 mm is also anacceptable support. The surface of the metal mesh, especially foraluminum meshes, was subjected to cleaning to remove dirt and oilsfollowed by washing the mesh in diluted sulfuric acid (10 to 20% H₂SO₄in DI water by volume) to remove resistive oxides and impurities. Thiscleaning improved nanofiber dispersion and adhesion. The depositedfibers may not be totally dried of the solvent used to dissolve thepolymers. In that state, the fibers adhere to the rigid mesh and aftertensioning after drying form a mesh-fiber structure beneficial to reducepressure drop and increase collection efficiency. Any number of metalsor metal alloys, with openings of various shapes (square, rectangle,circular, diamond, oblong and odd shaped), with openings ranging in sizefrom about 12.7 mm down to 1000 times the AFD can be used in theinvention.

Adhesion of the nanofibers or fibers to the support mesh can be improvedvia the application of an adhesive to the mesh directly prior toelectrospinning. The adhesive typically is a slow drying adhesivepermitting the adhesive to be tacky (i.e., adhesive) when electrospunfibers are deposited. Alternately, in another embodiment, the wires (orcomponents) of the mesh can be coated with a very thin layer of polymerthat has surface groups which interact (van der Waals, hydrogen-bond,dipole, electrostatic attraction, etc.) with the polymer fibers beingdeposited on the mesh. One example system is a thin coating ofpoly(glycidyl methacrylate) (PGMA) on nickel mesh with nanofibers ofpoly(methyl methacrylate) (PMMA) deposited on the coated mesh. Analternate embodiment of the invention uses cross linkable systems thatare polymerized after the fibers are deposited. Examples includechitosan nanofibers crosslinked with glutaraldehyde and polyvinylacetate crosslinked with borax; also, deposition of nanofibers onadhesives such as Norland's line of curable adhesives based onmercapto-ester compounds. These surface coatings increase adherence andadhesion of the nanofibers to the support.

The metal mesh can be replaced with metal foams such as ERG's Duocel™metal foams; for example, Aluminum Durocel with 20 pores per inch (PPI;alternately an average pore size of 1.27 mm). Foams can also be madewith copper, nickel, and various other metallic as well as polymericmaterials. Porosities ranging from 10 PPI (2.5 mm pores) to 40 PPI(0.064 mm pores) are acceptable for the invention.

The support mesh can be composed of a plastic that is conductive. Forexample polyester or nylon screen (or coarse nonwoven polymer mesh) iscoated with a conductive finish such as gold, palladium, or variousmetal alloys. The coating process can be achieved by any number ofestablished arts including vacuum deposition (e.g., sputter coating,evaporation deposition, and chemical vapor deposition), and chromeplating of plastics. Alternately, the mesh can be composed of conductiveplastic that obtains its conductivity via embedded conductive particles(carbon nanotubes, metals etc.); or, any method to make plastic meshconductive, semi-conductive, or electrostatic dissipating.

A nonwoven support that is conductive or made conductive (e.g., sputtercoating etc., as mentioned above) or moistening with a conductive fluidsuch as water can be used. The nonwoven support can make a largercontribution to the pressure drop but may be acceptable in certainapplications. In certain embodiments, use of woven scrim materials mayalso be acceptable for a base of a bioparticle collection medium.

The structure of the electric fields between the emitter and ground,which drives fiber deposition, are controlled, in part, by the design ofthe filter frame holder. Furthermore, the potential of the support meshcan be controlled by an electric field pulsation device (i.e., a voltagelimiter or discharge device or an electric field applicator device). Theelectric field pulsation device can be configured to pulse an electricfield at the collector at least once (or frequently) duringelectrospinning of the fibers to discharge charge accumulated on theelectrospun fibers.

Electrospun fibers carry charge to the mesh which is dischargedfrequently to ground by the voltage limiter device acting in thisexample as an electric field pulsation device. The resultant electricfield is oriented in the direction of the spinning fibers anddynamically modifies the structure of the electric field, therebyimparting improved fiber and mat properties (as measured by the FoM ofthe mat).

Filters having figures of merit greater than 20 kPa⁻¹ for average fiberdiameters of the nanofibers less than 200 nm and filters having a FoMgreater than 40 kPa⁻¹ for average fiber diameters of the nanofibers lessthan 100 nm have been also realized and are suitable for this invention.

The thickness of the fiber mat can vary from about 0.25 μm (250 nm) to500 μm or beyond if needed, where most filters had an average matthickness in the range of 2 to 5 microns. The average mat thicknessnumbers represent the average thickness of the total fiber mat in afilter. Alternately the mat thickness can be defined as layers of fiberswith the thickness including from 4 to 4000 layers where 4 to 400, or 5to 100, or 5 to 15 layers were typical in various embodiments.

The flexibility of electrospinning even allows mixed polymers such ascoaxial, mixed (blended) in same fiber, or deposited as layered orintermixed fibers. In addition to polymer chemistry and mixture ofpolymers, additives such as salts, proteins, and other materials can beincluded in the fibers via a variety of methods. These include directincorporation in the electrospinning solution, deposited or coated ontothe surface of the fibers during electrospinning using coaxial spinning,electrospin-spray or co-spinning U.S. Pat. No. 7,592,277 (the entirecontents of which are incorporated herein by reference). An alternativeto including additives as a part of the spinning process is to use apost-spinning process to coat the fibrous mat. The fibers can be coatedafter the fibers are formed via depositing the fibers into a liquid bathcontaining the additives or via dry or wet coating of the fiber matafter it is produced. A variety of these combinations is also possible.

Accordingly, the fibers and nanofibers produced by the inventioninclude, but are not limited to, acrylonitrile/butadiene copolymer,cellulose, cellulose acetate, chitosan, collagen, DNA, fibrinogen,fibronectin, nylon, poly(acrylic acid), poly(chloro styrene),poly(dimethyl siloxane), poly(ether imide), poly(ether sulfone),poly(ethyl acrylate), poly(ethyl vinyl acetate), poly(ethyl-co-vinylacetate), poly(ethylene oxide), poly(ethylene terephthalate),poly(lactic acid-co-glycolic acid), poly(methacrylic acid) salt,poly(methyl methacrylate), poly(methyl styrene), poly(styrene sulfonicacid) salt, poly(styrene sulfonyl fluoride),poly(styrene-co-acrylonitrile), poly(styrene-co-butadiene),poly(styrene-co-divinyl benzene), poly(vinyl acetate), poly(vinylalcohol), poly(vinyl chloride), poly(vinylidene fluoride),polyacrylamide, polyacrylonitrile, polyamide, polyaniline,polybenzimidazole, polycaprolactone, polycarbonate,poly(dimethylsiloxane-co-polyethyleneoxide), poly(etheretherketone),polyurethane, polyethyleneimine, polyimide, polyisoprene, polylactide,polypropylene, polystyrene, polysulfone, polyurethane,poly(vinylpyrrolidone), poly(2-hydroxy ethyl methacrylate) (PHEMA),gelatin, proteins, SEBS copolymer, silk (natural or syntheticallyderived), and styrene/isoprene copolymer.

Additionally, polymer blends can also be produced as long as the two ormore polymers are soluble in a common solvent or mixed solvent system. Afew examples would be: poly(vinylidene fluoride)-blend-poly(methylmethacrylate), polystyrene-blend-poly(vinylmethylether), poly(methylmethacrylate)-blend-poly(ethyleneoxide), poly(hydroxypropylmethacrylate)-blend poly(vinylpyrrolidone),poly(hydroxybutyrate)-blend-poly(ethylene oxide), proteinblend-polyethyleneoxide, polylactide-blend-polyvinylpyrrolidone,polystyrene-blend-polyester, polyester-blend-poly(hydroxyethylmethacrylate), poly(ethylene oxide)-blend poly(methyl methacrylate),poly(hydroxystyrene)-blend-poly(ethylene oxide)).

Other embodiments of the invention include the use of polymers that arepH and/or thermal responsive such that the fiber mat can later bemodified, respond to a change in environment, or easily dissolved.Example polymers include the commercial pH sensitive polymers know asEudragit polymers as well as copolymers of N-isopropyl acrylamide(NIPAM) and N-methyacryloy-L-Leucine (MALEU) or (N,N-dimethylamino)ethylmethacrylate (DMAEMA). A similar approach would be to use polymers thatare easily degraded with enzymes such as Chitosan which is degraded byChitosanase and cellulose which is degraded by α-cellulase. Combinationsof polymer systems could be used to tune the fiber filter mat propertiesto the particular application.

Other embodiments of the invention introduce an agent to the fibrousmatrix to reduce oxygen toxicity to bioparticles collected in thecollection medium. Such agents can be enzymes to reduce oxygen toxicityincluding catalase. Such agents can be fullerenes and modifiedfullerenes that have antioxidant properties. In general, substances withknown antioxidant properties can be added to the sample collectionmedium of the present invention to improve viability of the collectedbioparticles.

Accordingly, in one embodiment of the invention, fibrous mats can beelectrospun that provide favorable conditions for a variety oforganisms.

Working Examples of Fibrous Mats for Bioparticle Collection

A variety of polymer fiber mats were prepared for this invention usingelectrospinning. The polymer solution was prepared to a targetconcentration in a solvent system amenable to electrospinning. Thesolution was placed in a 3 ml syringe fitted with a 30 G blunt tippedneedle and placed into a controlled gas environment (see U.S. Pat. No.7,297,305, the entire contents of which are incorporated herein byreference) of carbon dioxide and a relative humidity (RH) of 30% to 35%.A high voltage was provided to the needle through an electrical lead tocreate a voltage gradient of about 1 kV/cm. The polymer flow rate wasdetermined by the electrospinning solution viscosity and ranged between0.05 to 0.1 ml/hr. The fibers are collected on a substrate dependingupon the manner in which the fiber mat was used.

For use as an impaction substrate, the collection substrate was aluminumfoil or Whatman filter paper; for use as a flow-though filter, thecollection substrate was a light weight nonwoven filter material such asFiberweb Reemay style 2011, which is a nonwoven filter material with abasis weight of 25.5 g/m² and an air permeability of 5,650 L/m²/s. Insome cases, the nonwoven backing is first coated with graphite, such asby spray painting using Aerosdag G to enhance fiber adhesion to thesubstrate. The electrospun fiber mat was then rinsed with filtered DIwater and dried in a sterile environment overnight to remove anyresidual solvents. In some cases, additives were coated onto thefinished mat in a post-processing step.

One fibrous material prepared was polysulfone (PSu; Udel P3500 LCD bySolvay Advanced Polymers) dissolved in dimethylacetimide (DMAc) at aconcentration of 21 wt %. 0.2 wt % tert-butyl ammonium chloride (TBAC)was then added to improve the electrospinning of the solution and thefinal fiber morphology. The solution was electrospun for 90 minutes at aflow rate of 0.05 ml/hr and voltage gradient of 1.6 kV/cm.

Another fibrous material prepared was polyurethane (PU; Pellethane by

Lubrizol) dissolved in dimethlyformamide (DMF) at 13 wt %. The solutionwas electrospun at 1.2 kV/cm and a flow rate of 0.1 ml/hr for 90minutes. More specifically, Pellethane 2103-90 AE Nat polyurethane (PU)made by Lubrizol, electrospun at about 13 wt % in dimethylformamide(DMF) to form micro and/or nanofibers was deposited on a backingmaterial. After the fibers are deposited on the backing, the fibrousmatrix was flushed with DI water and allowed to dry in a cleanenvironment.

The backing material can be any number of woven or nonwoven media suchas spunbound polypropylene. One example is Reemy spunbound polypropylenenonwoven made by Fiberweb. Media with air resistance of 500 CFM/ft2 to1,500 CFM/ft2 are useful but media with air resistance beyond this rangemay also be useful. In some cases, using a backing material that isconductive or static dissipating is advantageous. For example, anonwoven can be spray coated with graphite or coated with conductivematerial using liquid or gas based (e.g. chemical vapor deposition)techniques. Also materials known in the art that are static dissipatingthough any number of methods may be useful.

Another example of a nanofiber structure for viable collection andpreservation is PU electrospun onto Fiberweb Reemy 2250 that was coatedwith aerodag (graphite) before electrospinning. The PU fibers have anaverage fiber diameter of 320 nm, are free of beads, and form a nonwovenmat that is a few microns to 10 s of microns thick. Alternatively, thePU fibers can have an average fiber diameter in the range of 100 nm to280 nm. Alternatively, the fibrous matrix can have PU beads createdduring the electrospinning process that are 1.5 to 4.5 microns indiameter. In one embodiment, the average fiber diameter is 260 nm, andthe PU beads are about 4 microns in diameter. These nanofiber materialswith beads about 10 times to 20 times the size of the fibers providelower pressure drop.

FIGS. 11B-11E show SEMs of one embodiment of the nanofiber structureformed by deposition of PU on Fiberweb that was first coated withgraphite. In FIG. 11B, a section of backing material without nanofibersand a section with nanofibers are shown. The nanofibers are supported bythe nonwoven backing material. The nanofibers provide for collection ofthe bioparticles. In FIG. 11C, the beaded structure of the nanofibersand that they oriented between the supporting macrofibers of theconductive nonwoven they are deposited on is evident. This combinationof beading and fiber orientation provides a structure that has lowerpressure drop compared to other materials. In FIG. 11D, an edge (crosssectional) view of the nanofibers deposited upon the nonwoven backing isshown. The nanofiber layer is 10 s to 100 s of microns thick, but muchthinner than the supporting nonwoven material. In FIG. 11E a highmagnification SEM image is shown that indicates the fiber and beadstructure. The beads are typically oblong and are 10 to 20 times thesize of the fibers. The fibers are on the order of 220 to 280 nm whilethe beads are on the order of 3.8 to 4.8 microns.

Another fibrous matrix of this invention includes nylon fibers preparedfor example from a 15.3 wt % solution of nylon 6 (Sigma Aldrich)dissolved in formic acid. The solution was electrospun at a voltagegradient of 1.6 kV/cm and a flow rate of 0.05 ml/hr for 90 minutes.

Another fibrous matrix of this invention includes polycaprolactone (PCL;Sigma Aldrich, ca 43,000 M_(w)) fibers prepared from a mixed solventsystem. The solvent was composed of 80% methylene chloride and 20% DMF.PCL was dissolved in the mixed solvent to a concentration of 18 wt % andelectrospun at 1.1 kV/cm with a flow rate of 0.1 ml/hr for 90 minutes.

Another fibrous matrix of this invention includes polystyrene (PS; SigmaAldrich, ca 350,000 M_(w)) fibers prepared from 23 wt % PS in DMF andelectrospun for example at 1.2 kV/cm and a flow rate of 0.1 ml/hr for 90minutes.

In one embodiment, a fiber-facilitator is used. With a fiber-facilitator(e.g. high molecular weight PEO), nanocellulose and related cellulosicmaterials can be incorporated in the fibrous matrix to form fibers. Inother words, to assist in incorporating cellulose-based materials intofibers, a facilitator which is known in the art (e.g., high molecularweight PEO) to help in the formation of fibers, can be used.

One way according to this invention to impart improved viabilitymaintenance to the fibrous mat was to apply a solution containingadditives to the electrospun mat after it was made and rinsed with DIwater. The coated mat would then be allowed to dry in a sterileenvironment. For example, a solution containing a protein containingsolution tryptic soy broth can be applied to the mat and allowed to drybefore use.

Working Examples of Flow-Through Filters for Collecting Bioparticles

A sheet of nonwoven nanofiber filter media was prepared using PSu asdescribed above with the fibers being deposited on graphite coatedfiberweb support material. 37-mm circle filters were punched out of thesheet of nanofiber filter media and packaged into a standard 37-mm airsampling cassettes for testing.

FIG. 11F shows a nanofiber sampling filter in the 37 mm cassette formatrealized by this invention. The efficiency of the filter cassette wasmeasured with 300 nm KCl aerosol particles and was found to be >99.9%and have a pressure drop of 167 Pa for a face velocity of 5.3 cm/s. Asimilar filter cassette was exposed to bioaerosol, Bacillus globigii(Bg) or MS2, and analyzed via SEM and molecular biology techniques.Collection of Bg spores and MS2 particles was obtained. FIG. 12A showsSEM images of a collected Bg spore and what are likely MS2 virusparticles.

In one embodiment of the invention, the collection efficiency of thefiber mat structure (sampling filter) formed from a plurality of microor nanofibers can be >80% and more specifically >95% for particles 0.025μm to 10 μm in diameter for a flow rate of 25 L/min for a 25 mm samplingcassette. While at the same time, the pressure drop (air resistance) ofan unloaded fibrous sampling filter is less than 20 inches of water, andmore specifically less than 12 inches of water.

FIG. 12B compares the pressure drop curves (pressure drop versus facevelocity) for two common commercial air sampling filter materials withnanofiber filter media composed of PSU or PU deposited on graphitecoated fiberweb as described above. The structure formed by thelightweight backing material, the small fiber diameter, the partiallyoriented fibers, and beaded fibers (exemplified by PU nanofibers)provides for significant reduction in pressure drop. These significantlylower pressure drops of the nanofiber filters translate into advantagesfor both operation and equipment design. With a lower pressure dropacross the filter it is easier to maintain the target RH of the filterand therefore improve viability maintenance of the collectedbioparticles. Furthermore, with lower pressure drop the pumps andelectrical requirements for an air sampling device are smaller and morecost effective.

Remarkably, despite the efficiency >95% and pressure drop less than 12inches of water, the sampling filter is able to withstand loading withparticles until pressure drops greater than 80 inches of water, and evenas high as 100 inches of water.

The fibrous sampling filter described above is able to operate atcollection humidities ranging from 10% to 98% with no loss of filteringintegrity. However for viability considerations, it is preferablyoperated in the range of 70% to 85%.

While described here in relation to flow through sampling, these fibroussampling filters have application in the other sample collection devicesdescribed herein.

Bioparticle Collection, Testing, and Evaluation

Viable microorganisms were generated to test the viable collection ofthe samplers. Bioparticle generation was accomplished though the use ofa Collison nebulizer containing a suspension of microorganism. Themicroorganisms may be suspended in various nebulizing fluids dependingupon the organisms and the scenario being tested. Nebulizer fluids rangefrom sterile water to trypic soy broth with antifoam. The composition ofthe nebulizing fluids is often selected to simulate the conditions ofvarious bioparticles in the environment as the usual application forbioparticle samplers is to collect microorganism from the ambient orindoor air.

A recognized standard in the art of bioparticle collection is the AllGlass Impinger (AGI). The AGI is designed to draw aerosols through aninlet tube (e.g., a capillary tube) to form a jet of the aerosols to becaptured by a liquid medium of deionized water or impinger fluid. Thejet tip is typically positioned 30-mm above the base of the impinger.The AGI relies on the inertial impaction as a means for collection.However, loss of sampling liquid through evaporation andre-aerosolization of droplets containing virus often reduces collectionefficiency of liquid impingers.

The AGI provides collection into liquid for particles larger than about0.3 microns. Due to the wet collection, the majority of sampledorganisms are collected in a viable state. However, this method can onlybe operated for a short period of time, about 30 minutes. Yet, the AGIis a recognized collection system used as a point of comparison to thefibrous material collection devices of this invention. Andersenbiological impactors and the SKC biosampler were also suitable. Theliquid collection fluid is diluted (when necessary) and then analyzed.

Sampling of a controlled air stream containing an aerosol of a microbeat controlled concentration was conducted to compare sampling methods.In some cases an AGI is run for 30 minutes in parallel with the othersampling technology with the AGI being considered the “gold standard” tocompare viable sampling collection against. For example for a specifiedtest bacteria, 6% of AGI means that the method collected 6% of thecolony forming units per liter of sampled air that the AGI collected

Meanwhile, the fibrous material collection devices of this inventionwere suspended in sterile extraction fluid (e.g—water, phosphatebuffered saline, tryptic soy broth [TSB]), diluted and analyzed.

For comparison, the analysis for culturable organisms followed standardprocedures where an aliquot of collection fluid, extraction fluid, or adilution of either, is plated on microbiological media appropriate forthe microorganisms collected. The plated media were incubated at atemperature favorable for the microorganism growth and enumerated whencolonies (bacteria) or plaques (viruses) are countable.

Evaluation of Collection Via Condensation Growth Tube Condition

The method of conditioning the sampled aerosol with a CGT followed byimpaction onto the nanofiber mats of this invention was compared withCGT followed by impaction onto other substrates, with filtration airsampling using various filter materials, and the AGI.

In FIG. 13, two different aerosol challenges of Bg generated at twodifferent relative humidities (RH in the aerosol generation device) aresampled either with a CGT followed by impaction or via a 37 mm-diameterair sampling cassette containing different filter media. The filtrationsampling conducted here does not use additional conditioning (i.e. RHregulation) of the sampled air. The CGT used is an 8 L/min module andthe impaction substrate was either a 37-mm filter or impinger fluid. Theimpinger fluid can range in composition from sterile water to TSB withantifoam.

Filtration based air sampling using PSU nanofibers demonstrates betterviable collection than a CGT impacting on impinger fluid as shown inFIG. 13, Challenge level 1. The nanofiber filter is providing excellentcollection efficiency of the hardy Bg organism demonstrating that it isa viable method of air sampling for hardy organisms.

CGT conditioning of the air followed by impaction onto PSU nanofibersdemonstrates excellent viable collection compared to CGT with impactiononto impinger fluid or filtration based air sampling using the industrystandard Teflon filter (Teflo) as shown in FIG. 13, Challenge level 2.

Collection and preservation of vegetative organisms without growth issignificantly more challenging than spores (e.g. Bacillus spp.). A modelorganism for studying hazardous vegetative bacteria is Pseudomonasfluorescens. Results for collecting aerosolized P. fluorescens are shownin FIG. 14; sampling was done either with conditioning with the CGTfollowed by impaction onto various substrates or via air filtrationwithout any conditioning of the sampled air. Collection of the organismvia filtration without humidity control results in desiccation and theorganism is not able to survive. Viable collection of P. fluorescens viathe 8 L/min CGT impaction onto nanofiber substrates has beendemonstrated in FIG. 14. In this example two types of PSU nanofibersmats deposited on a sorbent backing were used, one without additives andone with dried TSB as an additive. The results showed that collection onnanofibers, as long as there is humidification at the collection point,worked well.

Another threat to health is viruses. Virus particles have the potentialto be much smaller than bacteria since the individual viruses can be assmall as 25 nm (0.025 μm). Collection and preservation of theseparticles can be quite challenging. MS2 bacteriophage was used as virussimulant and collected using nanofiber substrates as shown in FIG. 15;here again collection is done either with a CGT or filtration withouthumidity control. The results showed that nanofibers effectivelycollected the virus in both the cases of use with a CGT to condition thebioparticles prior to collection or as a sampling filter withoutconditioning of the sampled air. Results shown in FIG. 15 are reportedas plaque forming units (PFUs) per liter of air based on MS2 infectionof a lawn of E. coli.

To further assess the suitability of the CGT and the fiber technologiesof the invention for viable bioaerosol collect MS2 (approximately 0.02to 0.03 μm in diameter) and Bg (approximately 0.9 μm in diameter) werenebulized and collected. Collection was performed in parallel with an 8L/min CGT using a PSU nanofiber filter as an impaction substrate and anAGI. For the Bg experiments, an aerodynamic particle sizer (APS; TSIinc.) was also used.

FIG. 16 includes Table 2 presenting the results of the collection of BgCFUs and MS2 PFUs by CGT with nanofibers, the AGI, and the totalparticle number concentrations of Bg measured with the APS. The APSshowed consistent size distributions among runs, with a geometric meanaerodynamic diameter of D_(gm)=0.85 μm, equal to that expected for Bg.With a CGT sampler with impaction onto nanofibers, the number of CFUsmatched the concentrations measured with the APS. Moreover, as shown inTable 4, the standard deviation among runs, 18%, compared favorably tothe 7% variability in the APS counts. The AGI sometimes matched the APScounts and sometimes yielded lower values.

Collection of the virus simulant MS2 is compared to the AGI because itis one of the few bioaerosol samplers that are even capable ofefficiently collecting at sizes near 0.3 μm. With CGT and impaction ontonanofibers, the average concentration of culturable PFUs was 34% higherthan measured with the AGI. The coefficient of variation was 16%. Theabsolute viable collection efficiency is not yet known because the AGIis not efficient at collecting 0.3 μm and below, which is the size ofindividual or small clumps of viruses. In short, the CGT with impactiononto nanofibers is a better viable collector for virus particles thanthe AGI.

Conditioning of the sampled air with a CGT followed by impaction onto ananofiber mat provides viable collection for bacteria that is comparableto an AGI, However as moisture for the CGT is continually fed to thesystem, sampling is no longer limited to only 30 minutes required by theAGI. The CGT can perform very long term sampling limited only by thesize of the water reservoir feeding the CGT and by the time thebioparticles can survive on the collection substrate. This viabilityduring storage is further discussed below in the Section Storage ofCollected Bioparticles. Furthermore the CGT with impaction ontonanofibers has better viable collection of virus particles compared tothe AGI.

Evaluation of Collection Via Filtration and Humidity ControlledFiltration

The methods sampling of air using filtration and of adding moisture orcontrolling the humidity of the sampled air followed by filtration wereevaluated using aerosols of bioparticles and comparison with industrystandard filtration sampling methods or the AGI.

FIG. 17 includes Table 3 showing a comparison sampling the virus MS2using filtration without humidity control of the sampled air. Thenanofiber filter mats of the invention are compared to a standard Teflonfilter. Table 5 assesses both collection efficiency and viability. Thecollection efficiency of the polysulfone-based nanofiber filter wasnoticeably higher than the standard Teflon filter or the polystyrenenanofiber filter. (These results are for materials not optimized for aspecific microbe collection.)

For demonstration of humidity controlled filtration, a method ofcontrolling the humidity of the sampled air was constructed thatmeasured the RH immediately downstream of the sampling filter. The RH ofthe sampled air was controlled via mixing with a moist air stream,similar to that shown in FIG. 6. The moist airstream was generated bypassing clean, dry air through a bubbler containing deionized waterfollowed by HEPA filtration of the humidified air. The ratio of sampledair to wet air was set at the beginning of the experiment to providetarget RH at the filter. This ratio was noted and used to determine theactual volume of air with aerosol sampled from the test chambercontaining the aerosolized bioparticles. Various filter types includingnanofiber filters of this invention were used. In the art, gelatinfilters are recognized as having the best viable collection ofcommercially available materials. However, when used on their own, as istypical in the art, they too are very limited in the duration time forviable sampling of bioparticles, about 30 to 60 minutes.

Humidity controlled filtration was performed with the vegetativebacterium Serratia using nanofiber filters of this invention compared togelatin or Teflon filters. The nanofiber mats were punched into 25-mmcircular filters and placed into a standard 25-mm air sampling cassette.The gelatin and Teflon filters were used as received in a 37-mm airsampling cassette. Bioaerosol was sampled for 3 hours and the results ofCFUs of Serratia determined. FIG. 18 compares the results of thisexperiment. The nanofibers and gelatin perform better than the Teflonfilters. It should be noted that the gelatin filters have much higherpressure drop than the nanofiber filters, see FIG. 12B.

A similar experiment to that shown in FIG. 12B was attempted with longerterm sampling to compare gelatin and PU nanofiber filters for very longsampling times. However, the gelatin filters deteriorated sometime after3 hours of sampling and collected bioparticles could not be recovered.The PU nanofiber filters were found to withstand sampling times of morethan 32 hours of operation.

The impact of sampling face velocity as a function of filter materialfor RH controlled filtration was tested with 30 minute sampling ofSerratia as shown in FIG. 19. PU nanofibers and gelatin filters wereused with an RH of 75% and the face velocity varied. The PU nanofiberfilter is able to collect viable bioparticles at very high facevelocities that actually result in rupture of the gelatin filter. Asexpected the percent viable collected increases with face velocity asmore organism per area of filter are collected at the higher flow rates.Operation at high flow rates, as high as 100 L/min or even higher, isdesirable for air monitoring. For example release of biological weaponcould result in low concentrations of the organism in the air such thatsampling as much air as possible to generate as much collected organismas possible is desired (that is as much single collected for the eventas possible).

To demonstrate collection of a very fragile vegetative bacterium that isparticularly difficult to collect in viable form using filtration,Yersinia rohdei was collected using RH controlled filtration with PUnanofiber filters compared to an AGI. FIG. 20 shows that indeedcollection of even this very fragile organism is possible andrepeatable.

RH controlled filtration using nanofibers is an effective way to performlong term viable collection of bioparticles. Using the fibrous materialcollection devices of this invention provides viable collection similarto gelatin when both substrates are used in the same sampling system andsame RH for short periods of time and modest flow rates (face velocityless than 4,500 cm/min). However, the fibrous material collectiondevices of this invention provide several advantages over gelatinfilters: 1) filter pressure drop for nanofibers is much lower for thefibrous material collection devices than gelatin; 2) the robustness ofnanofibers in the fibrous material collection devices is much greaterthan gelatin. The fibrous material collection devices are able towithstand high pressure drops, in excess of 100 inch-H₂O, are able towithstand long term operation at >75% RH (e.g., more than 3 hrs, morethan 24 hrs). Furthermore, the nanofiber filters are free ofcontaminants that would interfere with or give false results formicrobiology assays.

Storage of Collected Viable Bioparticles

After collection, preserving the organisms is a significant challenge,particularly in the case where samples are not refrigerated.

In one embodiment of the invention, there is provided an automatedsystem for sequential particle collection and storage. In one embodimentof the invention, several days of samples are stored in a singlesampling cassette that will also contain an electronic tag indicatingindividual sample collection times, location, air sampling volumes, andquality assurance (QA) parameters (e.g., flows, operating temperatures,water levels, and other performance parameters). Another cassette caninclude consumables, including water for the CGT operation or provisionof elevated RH by other method and any supplies for sample collectionand preservation, depending upon the collection and handling schemeselected. In one embodiment, these cassettes would be exchanged in thefield during operation of the sample collector.

As a demonstration, organisms were inoculated via pipetting solutioncontaining microbes onto samples of various nanofiber or fibersubstrates and other substrates compatible with air samplers. Two typesof inoculations were done to simulate different environmentalconditions: “lightly protected” where a buffer with 0.25% TSB in sterilewater was used, and “well protected” where a full strength TSB bufferwas used. When an aerosol containing microorganisms or otherbioparticles is generated in the natural world, it always has othermaterials with it such as proteins, sugars, sputum, dirt etc thatprovides protection for the organisms. In a bioterrorist act, thebioparticles would be purposely mixed with protective materials likeprotein. The samples in this demonstration were stored at ambienttemperature (approximately 23° C.) in controlled RH static chambertests. The relative humidity RH in this demonstration was controlled viasaturated salt solutions in the sealed chambers. After storage, sampleswere extracted in buffer, such as TSB or phosphate buffered saline, andplatted on appropriate nutrient media and incubated and organismsenumerated.

FIGS. 21A and 21B include Tables 4 and 5 showing the results ofsurvivability of model organism on various materials. “Alive” means theorganism was detected via live culture techniques and “dead” means nonewere detected. A “D” and number indicated by days of live detection,e.g. D7 means live culture detected at day 7.

In another set of experiments where PU nanofiber filters of thisinvention were further studied, model organisms were inoculated viapipetting and samples stored under various conditions as shown in FIG.22. The log change from the day of inoculation (day 0) is reported.Storage of the slightly fragile Staphylococcus is possible under avariety of conditions. The very fragile organism Yersinia requiresstorage at cooled temperatures such as 4° C.

Storage of a range of bioparticles on nanofibers is possible. Fororganisms that are hardy to moderately hardy, storage under conditionsnot requiring cooling is possible. In some cases storage under humiditycontrolled conditions such as those provided by the RH control system ofthe sampled air are sufficient to preserve the collected bioparticles.In the case of fragile and very fragile organisms, cooled storage isrequired if viability maintenance for more than a day or two arerequired. As demonstrated, different collection substrates provideviable maintenance for different organisms. With the flexibility ofelectrospinning and other arts of making fibrous media a mixed polymerfiber environment can be created to provide for viable storage of abroad range of organisms not possible with a single traditionalmaterial.

Prior to the invention, the best filter for collection viability thatcurrently existed was a gelatin filter. However, the gelatin filter hasa number of problems including contamination and excessive drying duringlong term sampling without RH control, which both negatively impact thestorage viability. Another common filter medium is PTFE (Teflon) filter.Yet, the results above, especially for the design limiting organisms,show that both collection and storage viability and the collectionefficiency are enhanced for the fibrous material collection devices ofthis invention.

The above experiments with aerosolized bioparticles demonstrated thatnanofibers are good collectors of microbes (bioparticles). The aboveexperiments show that the selection of polymer and fiber structure isone element impacting viability and controllable by this invention. Theabove experiments show that preventing desiccation of bioparticles isimportant is one element impacting viability and controllable by thisinvention. The above experiments show that viability maintenance can beachieved through incorporation of viability sustaining additives,moisture, etc. and by keeping the fibrous media in an RH regulatedenvironment. These aspects are controllable by this invention.

Alternative Applications

In addition to the collection of bioparticles, the various embodimentsof the sampler can collect other aerosol particles of interest to thepublic health and air monitoring communities. The sampler may be usedoutdoors to sample ambient air or for sampling indoors in buildings,arenas, or transportation facilities. These filters also offer anadvantage because of their semi-transparency for black carbon absorptionanalysis and low levels of analysis interfering metals.

In these applications, the ambient air samples will contain black carbonor soot from combustion sources, industrial pollution, particles fromatmospheric reactions, particles re-suspended from soil and pavements,ocean generated particles and pollen, all of which can be collected bythe nanofiber collection devices of this invention. When used for indoorapplications, it is expected that the occupant generated particles suchas skin cells and residue of personal care products, dust and fibersresuspended from carpets and floors, smoking, and particles introducedfrom appliances such as electrical motors and heaters or furnaces, andbiological material such as toxins and plant or animal debris, all ofwhich can be collected by the nanofiber collection devices of thisinvention. Aerosol particles collected in the nanofiber filters could bemeasured by light absorption or reflectance, microscopy, weighing andchemical analysis.

While described above with respect to aerosol sampling, the nanofibermedia and aspects contributing to viable collection and maintenance ofbioaerosols have applications in the sampling of bioparticles andorganisms from surfaces and from water. For example, a wipe or brush orother sample collection device containing the nano or microfibermaterial described above that provides sample collection and helpsviability maintenance could be used to collect bioparticles from akeyboard, lab bench, furniture, vehicle interior, etc. The wipe or brushor other collection device can then be transported with the viabilitymaintenance materials to a laboratory for analysis.

In one embodiment of the invention, the sample collection device can bein the form of wipes, brushes, swabs, sorbent pads, liquid filters, airfilters, and/or similar devices for sampling air, liquids, or surfaces.Applications include forensics, regulatory compliance, surveillance,etc.

For embodiments of the invention where the nanofiber material is used tocollect microbes without incorporation of mechanisms to control thehumidity of inlet air, a container can be used that provides a humiditycontrolled environment. For example a wipe composed of a plurality offibers with viability enhancing properties for use in evidencecollection. In one embodiment that nanofiber wipe is stored in a sterilecontainer with humidity regulation. In other embodiments the wipe isstored sterile but humidity is not required prior to use. The nanofiberwipe is then used to collect a sample and is placed in the containerwhere the container provides a favorable RH environment for viabilitymaintenance during transport to the laboratory for analysis. Thecontainer and wipe thus constitute a sample collection device thatprovides for viability maintenance.

The samples can then be stored in a sample storage device which canincorporate a moisture providing material or mechanism such as ahydrogel, water saturated salt solution, water reservoir separated fromthe nanofibers via a moisture permeable membrane providing watertransport, or a water reservoir connected to the part of the containerholding the nanofibers via a wick. Examples of these are shown in FIGS.23 and 24. FIG. 23 is a schematic depiction of a sample storage deviceincorporating a moisture providing material. FIG. 24 is a schematicdepiction of a sample storage device incorporating a moisture providingmechanism (e.g., a wick and a water reservoir). The moisture providingmaterial and the moisture providing mechanism can further providenutrient or antioxidants, as described above.

Generalized Aspects of the Invention

In one aspect of the invention, a collection device includes a pluralityof fibers formed into a fiber mat. The fiber mat is configured tocollect and maintain the viability of microbes and/or bioparticles. Thefibrous filter can be configured in any manner used in airsampling/aerosol collection using a flow-through filter. In one aspectof the invention, the fibers are configured as an impaction substrate tocollect and maintain microbes and/or bioparticles for use in airsampling/aerosol collection using the method of impaction. The fibrousfilter can be configured in any manner used for swabbing or the wipingof surfaces or the sampling of bioparticles in liquids.

In one aspect of the invention, there is provided a bioparticlecollection device including a collection medium including a plurality offibers formed into a fiber mat and configured to collect bioparticlesthereon, and including a viability enhancing material provider disposedin a vicinity of the plurality of fibers and configured to provide aviability enhancing material to the collected bioparticles to maintainviability of the bioparticles collected by the fiber mat. In this aspectof the invention, the viability enhancing material may or may not be asubset of the plurality of fibers.

In one aspect of the invention, the filter or impaction substrateincludes a fibrous mat configured in terms of structure, surfacechemistry, and additives to provide enhanced support of viabilitymaintenance of the bioparticles collected.

In one aspect of the invention, a filtration or impaction device foraerosol collection includes a fibrous mat and in conjunction with amechanism or method for conditioning the moisture content of the airentering the air sampling device to a value that provides enhancedcollection and maintenance of the bioparticles collected.

In one aspect of the invention, a device including a condensation growthtube is used to increase aerosol particle size with condensation ofwater moisture followed by impaction onto a fibrous substrate or asubsequent filtration mechanism. The fibrous substrate and/or thesubsequent filtration mechanism provide a collection mechanism ofbioparticles and provide a mechanism for maintenance of viability of thecollected bioparticles.

In one aspect of the invention, the aerosol is exposed to the vapor or aworking fluid (such as for example water and other fluids that arebiocompatible, possibly including silicone fluids) in a saturationchamber. Subsequently, vapor condensation onto particles is induced byeither adiabatic expansion or cooling in the condensing chamber, or bymixing with a cooler airflow. The enlarged particles are subsequentlycollected via impaction or filtration on a nanofiber or fiber material.

In one aspect of the invention, a fibrous mat is configured to provideenhanced recovery of the collected material. Enhanced recoveryincludes 1) recovery of the particles such that the extraction proceduredoes not decrease their viability; 2) a collection and extraction whichdoes not prevent or impede subsequent analysis such as live culture,PCR-based techniques, or any other chemical or physical analysis of thecollected material/organisms; and 3) enhanced release of the collectedmaterial through dissolution of the fibrous material using selectsolvents and/or processing conditions.

In one aspect of the invention, the fibers in the fibrous material orthe fiber mat have an average fiber diameter of less than 10 μm, or lessthan 1 μm, or less than 500 nm, or less than 300 nm, or less than 200nm, or less than 100 nm.

In another aspect of the invention, there is provided a method forcollecting aerosols. This method includes entraining particles in a gasstream, saturating the particles in the gas stream with a solvent, andcollecting the saturated aerosol particles by a collection medium. Thecollection medium includes a plurality of fibers formed into a fiber matincluding and a viability enhancing material provider disposed in avicinity of the plurality of fibers and configured to provide aviability enhancing material to the collected bioparticles to maintainviability of the bioparticles collected by the fiber mat.

This method also can inject the viability enhancing material into thecollection medium prior to collecting the aerosol particles. Theviability enhancing material injected can be at least one of water,proteins, carbohydrates, sugars, salts, phosphate buffered saline, andtryptic soy broth. This method also can inject the viability enhancingmaterial (such as those listed above) into the collection medium duringthe collecting of the aerosol particles. This method also can injectantioxidants such as for example nitrous oxide (NO) into the collectionmedium.

This method also can introduce an agent to reduce oxygen toxicity to thebioparticles collected in the collection medium. Such an agent caninclude enzymes or fullerenes to reduce oxygen toxicity.

In another aspect of the invention, there is provided a bioparticlecollection device including a collection medium including a plurality offibers formed into a fiber mat and an osmotic material disposed incontact with the plurality of fibers. The osmotic material can be aviability enhancing material configured to maintain viability ofbioparticles collected by the fiber mat. The osmotic material can be awater-regulating material configured to provide water to the fibers. Theosmotic material can constitute a nutrient supply providing nutrients tosupport biological viability of biomaterial collected in the filtrationmedium. The nutrient supply can be at least one of water, proteins,sugars, carbohydrates, salts, phosphate buffered saline, and tryptic soybroth.

The collection medium and the viability enhancing material can bedisposed in one of an air filter, a wipe, a brush, a swab, a sorbentpad, or a liquid filter. The fibers can be made of materials which aredissolvable in a bio-compatible solvent.

The collection medium can include a support (e.g., a rigid support)supporting the collection medium. The support can be one of a filter, aplastic foam, a metallic foam, a semi-conductive foam, a woven material,a nonwoven material, a plastic screen, a textile, and a high efficiencyparticulate air (HEPA) filter medium.

In another aspect of the invention, there is provided an aerosolcollection system including an aerosol pumping device configured toentrain particles in a gas stream, an aerosol saturation deviceconfigured to saturate the particles in the gas stream with abiocompatible liquid, and an aerosol collection medium downstream fromthe aerosol saturation device. The aerosol collection medium includes aplurality of fibers formed into a fiber mat for collection of thesaturated aerosol particles, and an osmotic material disposed in contactwith the plurality of fibers.

The aerosol collection system can include a humidity control deviceconfigured to maintain the collection medium at a relative humidity from50 to 100%, or at a relative humidity from 65 to 85%, or at a relativehumidity from 75 to 81%.

The aerosol collection medium in this aspect of the invention can be atleast one of a flow-through or an impaction device. The osmotic materialin this aspect of the invention can be a viability enhancing materialconfigured to maintain viability of bioparticles collected by the fibermat. The osmotic material in this aspect of the invention can be awater-regulating material configured to provide water to the fibers. Theosmotic material in this aspect of the invention can be a nutrientsupply providing nutrients to support biological viability ofbiomaterial collected in the filtration medium. The nutrient supply inthis aspect of the invention can be a supply of at least one ofproteins, sugars, and salts.

The fibers in this aspect of the invention can be nanofibers, can beformed of materials dissolvable in a bio-compatible solvent. A support(rigid or not) can be used to support the collection medium. The supportin this aspect of the invention can be at least one of a filter, aplastic foam, a metallic foam, a semi-conductive foam, a woven material,a nonwoven material, a plastic screen, a textile, and a high efficiencyparticulate air (HEPA) filter medium.

In another aspect of the invention, there is provided a method forcollecting aerosols. The method included entraining particles in an gasstream, saturating the particles in the gas stream with a biocompatibleliquid, and collecting the saturated aerosol particles by a collectionmedium including a plurality of fibers formed into a fiber mat includingand an osmotic material disposed in contact with the plurality offibers.

This method also can inject the viability enhancing material into thecollection medium prior to collecting the aerosol particles. Theviability enhancing material injected can be at least one of water,proteins, carbohydrates, sugars, salts, phosphate buffered saline, andtryptic soy broth. This method also can inject the viability enhancingmaterial (such as those listed above) into the collection medium duringthe collecting of the aerosol particles. This method also can injectantioxidants such as for example nitrous oxide (NO) into the collectionmedium.

This method also can introduce an agent to reduce oxygen toxicity to thebioparticles collected in the collection medium. Such an agent caninclude enzymes or fullerenes to reduce oxygen toxicity.

In another aspect of the invention, there is provided a bioparticlecollection device including a collection medium including a plurality offibers formed into a fiber mat and an osmotic material disposed incontact with the plurality of fibers. The osmotic material can be aviability enhancing material configured to maintain viability ofbioparticles collected by the fiber mat. The osmotic material can be awater-regulating material configured to provide water to the fibers. Theosmotic material can constitute a nutrient supply providing nutrients tosupport biological viability of biomaterial collected in the filtrationmedium. The nutrient supply can be at least one of water, proteins,sugars, carbohydrates, salts, phosphate buffered saline, and tryptic soybroth.

The collection medium and the viability enhancing material can bedisposed in one of an air filter, a wipe, a brush, a swab, a sorbentpad, or a liquid filter. The fibers can be made of materials which aredissolvable in a bio-compatible solvent.

The bioparticle collection device can include a support (e.g., a rigidsupport) supporting the collection medium. The support can be one of afilter, a plastic foam, a metallic foam, a semi-conductive foam, a wovenmaterial, a nonwoven material, a plastic screen, a textile, and a highefficiency particulate air (HEPA) filter medium.

Numerous modifications and variations of the invention are possible inlight of the above teachings. It is therefore to be understood thatwithin the scope of the appended claims, the invention may be practicedotherwise than as specifically described herein.

1. A bioparticle collection device comprising: a collection mediumincluding a plurality of fibers formed into a fiber mat and configuredto collect bioparticles thereon; and a viability enhancing materialprovider disposed in a vicinity of the plurality of fibers andconfigured to provide a viability enhancing material to the collectedbioparticles to maintain viability of the bioparticles collected by thefiber mat.
 2. The device of claim 1, wherein the viability enhancingmaterial provider comprises an osmotic material disposed in contact withthe plurality of fibers and configured to maintain a relative humiditysuitable for said viability of bioparticles.
 3. The device of claim 2,wherein the osmotic material comprises a water-regulating materialconfigured to provide water to the fibers.
 4. The device of claim 1,wherein the osmotic material comprises a nutrient supply providingnutrients to support biological viability of biomaterial collected inthe fiber mat.
 5. The device of claim 4, wherein the nutrient supplycomprises a supply of at least one of water, proteins, carbohydrates,sugars, salts, phosphate buffered saline, and tryptic soy broth.
 6. Thedevice of claim 1, wherein the collection medium and the viabilityenhancing material are disposed in one of an air filter, a wipe, abrush, a swab, a sorbent pad, or a liquid filter.
 7. The device of claim1, wherein the plurality of fibers comprise materials dissolvable in abio-compatible solvent.
 8. The device of claim 1, further comprising asupport supporting the collection medium.
 9. The device of claim 8,wherein the support comprises: at least one of a filter, a plastic foam,a metallic foam, a semi-conductive foam, a woven material, a nonwovenmaterial, a plastic screen, a textile, and a high efficiency particulateair (HEPA) filter medium.
 10. The device of claim 1, wherein theviability enhancing material provider comprises at least a part of theplurality of fibers.
 11. The device of claim 1, wherein the viabilityenhancing material provider comprises a material different from theplurality of fibers.
 12. The device of claim 1, wherein the fibers havean average fiber diameter of less than 10 microns.
 13. The device ofclaim 1, wherein the fiber mat comprises between 4 to 4000 layers of thefibers.
 14. The device of claim 1, wherein the fiber mat comprises athickness between 0.25 and 500 μm.
 15. The device of claim 1, whereinthe fibers comprise a pH responsive material.
 16. The device of claim 1,wherein the fibers comprise at least one of acrylonitrile/butadienecopolymer, cellulose, cellulose acetate, chitosan, collagen, DNA,fibrinogen, fibronectin, nylon, poly(acrylic acid), poly(chlorostyrene), poly(dimethyl siloxane), poly(ether imide), poly(ethersulfone), poly(ethyl acrylate), poly(ethyl vinyl acetate),poly(ethyl-co-vinyl acetate), poly(ethylene oxide), poly(ethyleneterephthalate), poly(lactic acid-co-glycolic acid), poly(methacrylicacid) salt, poly(methyl methacrylate), poly(methyl styrene),poly(styrene sulfonic acid) salt, poly(styrene sulfonyl fluoride),poly(styrene-co-acrylonitrile), poly(styrene-co-butadiene),poly(styrene-co-divinyl benzene), poly(vinyl acetate), poly(vinylalcohol), poly(vinyl chloride), poly(vinylidene fluoride),polyacrylamide, polyacrylonitrile, polyamide, polyaniline,polybenzimidazole, polycaprolactone, polycarbonate, polyurethanes,poly(dimethylsiloxane-co-polyethyleneoxide), poly(etheretherketone),polyethylene, polyethyleneimine, polyimide, polyisoprene, polylactide,polypropylene, polystyrene, polysulfone, polyurethane,poly(vinylpyrrolidone), poly(2-hydroxy ethyl methacrylate) (PHEMA),gelatin, proteins, SEBS copolymer, silk (natural or syntheticallyderived), styrene/isoprene copolymer, and polymer blends including atleast one of poly(vinylidene fluoride)-blend-poly(methyl methacrylate),polystyrene-blend-poly(vinylmethylether), poly(methylmethacrylate)-blend-poly(ethyleneoxide), poly(hydroxypropylmethacrylate)-blend poly(vinylpyrrolidone),poly(hydroxybutyrate)-blend-poly(ethylene oxide), proteinblend-polyethyleneoxide, polylactide-blend-polyvinylpyrrolidone,polystyrene-blend-polyester, polyester-blend-poly(hydroxyethylmethacrylate), poly(ethylene oxide)-blend poly(methyl methacrylate), andpoly(hydroxystyrene)-blend-poly(ethylene oxide)).
 17. The device ofclaim 1, wherein the fibers comprise at least one of polyethylene oxide(PEO), poly(2-hydroxy ethyl methacrylate) (PHEMA), gelatine, andpoly(vinyl alcohol), cellulose acetate, chitosan, poly(lactic acid),collagen, poly caprolactone, poly(ethylene glycol) methyl ether,polystyrene.
 18. The device of claim 1, wherein the viability enhancingmaterial provider is intermixed with the plurality of fibers.
 19. Thedevice of claim 1, wherein the viability enhancing material comprises alayer of biomaterial separated from the plurality of fibers.
 20. Thedevice of claim 1, wherein the viability enhancing material providercomprises at least one of a polycarbonate, Eudragit polymers, copolymersof N-isopropyl acrylamide (NIPAM) and N-methyacryloy-L-Leucine (MALEU)or (N,N-dimethylamino)ethyl methacrylate (DMAEMA), cellulose, anddegraded cellulose.
 21. An aerosol collection system comprising: anaerosol pumping device configured to entrain particles in an gas stream;an aerosol saturation device configured to saturate the particles in thegas stream with a biocompatible liquid; and an aerosol collection mediumdownstream from the aerosol saturation device and including, a pluralityof fibers formed into a fiber mat for collection of the saturatedaerosol particles, and a viability enhancing material provider disposedin a vicinity of the plurality of fibers and configured to provide aviability enhancing material to the collected bioparticles to maintainviability of the bioparticles collected by the fiber mat.
 22. The systemof claim 21, further comprising a humidity control device configured tomaintain the collection medium at a relative humidity from 50 to 100%.23. The system of claim 21, further comprising a humidity control deviceconfigured to maintain the collection medium at a relative humidity from65 to 85%.
 24. The system of claim 21, further comprising a humiditycontrol device configured to maintain the collection medium at arelative humidity from 75 to 81%.
 25. The system of claim 21, whereinthe aerosol collection medium comprise at least one of a flow-through oran impaction device.
 26. The system of claim 21, wherein the viabilityenhancing material provider comprises an osmotic material disposed incontact with the plurality of fibers and configured to maintain arelative humidity suitable for said viability of bioparticles.
 27. Thesystem of claim 26, wherein the osmotic material comprises awater-regulating material configured to provide water to the fibers. 28.The system of claim 26, wherein the osmotic material comprises anutrient supply providing nutrients to support biological viability ofbiomaterial collected in the filtration medium.
 29. The system of claim28, wherein the nutrient supply comprises a supply of at least one ofproteins, sugars, and salts.
 30. The system of claim 21, wherein theplurality of fibers comprise nanofibers.
 31. The system of claim 21,wherein the plurality of fibers comprise materials dissolvable in abio-compatible solvent.
 32. The system of claim 31, further comprising asupport supporting the collection medium.
 33. The system of claim 32,wherein the support comprises: at least one of a filter, a plastic foam,a metallic foam, a semi-conductive foam, a woven material, a nonwovenmaterial, a plastic screen, a textile, and a high efficiency particulateair (HEPA) filter medium.
 34. The system of claim 21, wherein theviability enhancing material provider comprises at least a part of theplurality of fibers.
 35. The system of claim 21, wherein the viabilityenhancing material provider comprises a material different from theplurality of fibers.
 36. The system of claim 21, wherein the fibers havean average fiber diameter of less than 10 microns.
 37. The system ofclaim 21, wherein the fiber mat comprises a thickness between 0.25 and500 μm.
 38. The system of claim 21, wherein the fibers comprise a pHsensitive material.
 39. The system of claim 21, wherein the viabilityenhancing material provider is intermixed with the plurality of fibers.40. The system of claim 21, wherein the viability enhancing materialcomprises a separate layer from a layer including the plurality offibers.
 41. The system of claim 21, wherein the aerosol collectionmedium comprises at least one of a flow-through medium or an impactionmedium.
 42. A method for collecting aerosols, comprising: entrainingparticles in an gas stream; saturating the particles in the gas streamwith a biocompatible liquid; and collecting the saturated aerosolparticles by a collection medium including a plurality of fibers formedinto a fiber mat including and a viability enhancing material providerdisposed in a vicinity of the plurality of fibers and configured toprovide a viability enhancing material to the collected bioparticles tomaintain viability of the bioparticles collected by the fiber mat. 43.The method of claim 42, further comprising injecting the viabilityenhancing material into the collection medium prior to collecting theaerosol particles.
 44. The method of claim 43, wherein the injectingcomprises injecting at least one of water, proteins, carbohydrates,sugars, salts, phosphate buffered saline, and tryptic soy broth.
 45. Themethod of claim 42, further comprising injecting the viability enhancingmaterial into the collection medium during the collecting of the aerosolparticles.
 46. The method of claim 45, wherein the injecting comprisesinjecting at least one of water, proteins, carbohydrates, sugars, salts,phosphate buffered saline, and tryptic soy broth.
 47. The method ofclaim 42, further comprising introducing antioxidant gases into thecollection medium.
 48. A bioparticle collection device comprising: acollection medium including a plurality of fibers formed into a fibermat; and an osmotic material disposed in contact with the plurality offibers.
 49. An aerosol collection system comprising: an aerosol pumpingdevice configured to entrain particles in an gas stream; an aerosolsaturation device configured to saturate the particles in the gas streamwith a biocompatible liquid; and an aerosol collection medium downstreamfrom the aerosol saturation device and including, a plurality of fibersformed into a fiber mat for collection of the saturated aerosolparticles, and an osmotic material disposed in contact with theplurality of fibers.
 50. A method for collecting aerosols, comprising:entraining particles in an gas stream; saturating the particles in thegas stream with a biocompatible liquid; and collecting the saturatedaerosol particles by a collection medium including a plurality of fibersformed into a fiber mat including and an osmotic material disposed incontact with the plurality of fibers.